George G. Harrigan

Natural variation in crop composition and the impact of transgenesis

volume 28 number 5 mAY 2010 nature biotechnology These, and other studies (e.g., refs. 3–5), have also suggested a high degree of natural variability inherent to crop biochemical and metabolite composition. It is therefore reasonable to ask if changes in composition associated with modern transgenic breeding practices are different in scope from those attributable to natural genotypic and environmentally mediated variation. We reasoned that a systematic analysis encompassing published compositional data generated under OECD guidelines on several GM products grown in a range of geographies, under different regional agronomic practices and over multiple seasons would provide an effective overview of the relative impacts of transgenesis-derived agronomic traits with natural variation on crop composition. GM corn and GM soybean now represent 30.0% and 53%, respectively, of global production6. Our analysis therefore evaluated compositional data reported on grain and seed harvested from different GM corn and GM soybean products as these now represent a significant percentage of global production of these crops as well as provide an abundance of compositional data from diverse climates and growing regions. The high-quality compositional data generated according to principles outlined in the Organization for Economic Cooperation and Development (OECD; Paris) consensus documents2 are available. On a product-byproduct basis, compositional equivalence of GM crops and their conventional comparators has been demonstrated in potato, cotton, soybean, corn, rice, wheat and alfalfa (for a list of references describing compositional and omics comparisons of GM and non-GM comparators, see Supplementary References). In addition to the compositional studies conducted within regulatory programs, biochemical studies on GM crops have been extensively pursued by public and private research sectors. Although there are complexities in the interpretation of modern profiling technologies, and no standardized framework for comparisons, the lack of variation between GM crops and their conventional comparators at the transcriptomic, proteomic and metabolomic level has been independently corroborated. These profiling evaluations extend to a wide range of plants including wheat, potato, soybean, rice, tomato, tobacco, Arabidopsis and Gerbera (see Supplementary References). To the Editor: Compositional equivalence of crops improved through biotech-derived transgenic, or genetically modified (GM), traits and their conventional (non-GM) comparators is an important criterion in breeding as well as a key aspect of risk assessments of commercial candidates. We present here an analysis evaluated from compositional data on GM corn and GM soybean varieties grown across a range of geographies and growing seasons with the aim of not only assessing the relative impact of transgene insertion on compositional variation in comparison with the effect of environmental factors but also reviewing the implications of these results on the safety assessment process. Specifically, our analysis includes evaluation of seven GM crop varieties from a total of nine countries and eleven growing seasons. On the basis of our data, we conclude that compositional differences between GM varieties and their conventional comparators were encompassed within the natural variability of the conventional crop and that the composition of GM and conventional crops cannot be disaggregated. Plant breeding programs expect to either maintain compositional quality during enhancement of other agronomic traits or improve crop compositional quality through intended changes in the levels of key nutrients or antinutrients. Over the past two decades, one of the most successful approaches to enhancing agronomic traits in crops is the insertion of trait-encoding genes using the techniques of modern biotech. Compositional equivalence between GM crops and conventional (non-GM) comparators is an important breeding goal but is also often considered to provide an “equal or increased assurance of the safety of foods derived from genetically modified plants”1. Comparative compositional studies are therefore included as a significant component of risk assessments of new GM crops. As a consequence, a large body of Natural variation in crop composition and the impact of transgenesis

Impact of Genetics and Environment on the Metabolite Composition of Maize Grain

This study sought to assess genetic and environmental impacts on the metabolite composition of maize grain. Gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) measured 119 identified metabolites including free amino acids, free fatty acids, sugars, organic acids, and other small molecules in a range of hybrids derived from 48 inbred lines crossed against two different tester lines (from the C103 and Iodent heterotic groups) and grown at three locations in Iowa. It was reasoned that expanded metabolite coverage would contribute to a comprehensive evaluation of the grain metabolome, its degree of variability, and, in principle, its relationship to other compositional and agronomic features. The metabolic profiling results established that the small molecule metabolite pool is highly dependent on genotypic variation and that levels of certain metabolite classes may have an inverse genotypic relationship to each other. Different metabolic phenotypes were clearly associated with the two distinct tester populations. Overall, grain from the C103 lines contained higher levels of free fatty acids and organic acids, whereas grain from the Iodent lines were associated with higher levels of amino acids and carbohydrates. In addition, the fold-range of genotype mean values [composed of six samples each (two tester crosses per inbred x three field sites)] for identified metabolites ranged from approximately 1.5- to 93-fold. Interestingly, some grain metabolites showed a non-normal distribution over the entire corn population, which could, at least in part, be attributed to large differences in metabolite values within specific inbred crosses relative to other inbred sets. This study suggests a potential role for metabolic profiling in assisting the process of selecting elite germplasm in biotechnology development, or marker-assisted breeding.

Metabolomics, metabolic diversity and genetic variation in crops

The metabolome represents a critical aspect of a plant’s physiology, growth characteristics and, ultimately, economic value. Metabolic changes underpin plant development and responses to applied stresses, whilst quality traits in many important crops and ornamental plants are dependent on metabolic composition. It is also frequently reasoned that metabolic information reflects biological endpoints more accurately than transcript or protein analysis. As such, the science of metabolomics has proven to be of increasing popularity in assessing genotypic and phenotypic diversity in plants, in defining biochemical changes associated with developmental changes during plant growth and, increasingly, in compositional comparisons. The postulated value of this metabolic information resides primarily in its potential to support breeding and selection of novel yield-enhanced and nutritionally improved crops. Plants display remarkable genetic plasticity which has served to facilitate the development of an extraordinary range of genetically distinct and metabolically diverse cultivars for any given plant species. Despite concerns regarding potential loss of genetic diversity through domestication, genetic resources still exist through wild and exotic germplasms. Additionally, emerging biotechnological approaches such as RNAi silencing and the transgenic modification of regulatory genes offer new and fascinating opportunities for enhancing diversity and eliciting trait improvements. This review is intended to promote the view that metabolomics, through comparative assessments of metabolic diversity in domesticated and non-domesticated plants, and through evaluations of the compositional impact of metabolic engineering efforts can support breeding programmes designed to elicit trait improvements.

Compositions of Forage and Seed from Second-Generation Glyphosate-Tolerant Soybean MON 89788 and Insect-Protected Soybean MON 87701 from Brazil Are Equivalent to Those of Conventional Soybean (Glycine max)

Brazil has become one of the largest soybean producers. Two Monsanto Co. biotechnology-derived soybean products are designed to offer benefits in weed and pest management. These are second-generation glyphosate-tolerant soybean, MON 89788, and insect-protected soybean, MON 87701. The second-generation glyphosate-tolerant soybean product, MON 89788, contains the 5-enolpyruvylshikimate-3-phosphate synthase gene derived from Agrobacterium sp. strain CP4 (cp4 epsps). MON 87701 contains the cry1Ac gene and expression of the Cry1Ac protein providing protection from feeding damage caused by certain lepidopteran insect pests. The purpose of this assessment was to determine whether the compositions of seed and forage of MON 89788 and MON 87701 are comparable to those of conventional soybean grown in two geographically and climatically distinct regions in multiple replicated sites in Brazil during the 2007-2008 growing season. Overall, results demonstrated that the seed and forage of MON 89788 and MON 87701 are compositionally equivalent to those of conventional soybean. Strikingly, the results also showed that differences in mean component values of forage and seed from the two controls grown in the different geographical regions were generally greater than that observed in test and control comparisons. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) of compositional data generated on MON 89788, MON 87701, and their respective region-specific controls provide a graphical illustration of how natural variation contributes more than biotechnology-driven genetic modification to compositional variability in soybean. Levels of isoflavones and fatty acids were particularly variable.

Compositions of Seed, Forage, and Processed Fractions from Insect-Protected Soybean MON 87701 Are Equivalent to Those of Conventional Soybean

Monsanto Co. has developed biotechnology-derived, insect-protected soybean MON 87701 that produces the Cry1Ac insecticidal crystal (delta-endotoxin) protein derived from Bacillus thuringiensis (Bt) subsp. kurstaki. Cry1Ac provides protection from feeding damage caused by certain targeted lepidopteran pests. The purpose of this work was to assess whether the compositions of seed, forage, and processed fractions (meal, oil, protein isolate, and lecithin) of MON 87701 are comparable to those of conventional soybean. Compositional analyses were conducted on seed and forage tissues harvested from MON 87701 and conventional soybean grown in multiple replicated sites in the United States during the 2007 growing season and in Argentina during the 2007-2008 growing season. Seed, forage, and processed fractions from conventional soybean varieties currently in the marketplace were included in the analyses to establish a range of natural variability for each compositional component; the range of variability was defined by a 99% tolerance interval. Additional seed was collected from soybean grown in a separate U.S. production during the 2007 season. This seed and processed fractions (meal, oil, protein isolate, and crude lecithin) derived from it were also subjected to compositional analyses. Forage samples were analyzed for levels of proximates (ash, fat, moisture, and protein), carbohydrates by calculation, and fiber. Seed samples were analyzed for proximates, carbohydrates by calculation, fiber, amino acids, fatty acids, antinutrients, and vitamin E. Toasted, defatted (TD) meal was analyzed for proximates, fiber, amino acids, and antinutrients. Refined, bleached, and deodorized (RBD) oil was analyzed for fatty acids and vitamin E. Protein isolate was analyzed for amino acids and moisture. Crude lecithin was analyzed for phosphatides. Overall, results demonstrated that the seed, forage, and processed fractions of MON 87701 are compositionally equivalent to those of conventional soybean.

The forage and grain of MON 87460, a drought-tolerant corn hybrid, are compositionally equivalent to that of conventional corn.

MON 87460 contains a gene that expresses cold shock protein B (CSPB) from Bacillus subtilis. Expression of this gene confers a yield advantage when yield is limited by water availability. Compositional analyses of MON 87460 and a conventional corn variety with similar background genetics were conducted on forage and grain harvested from multiple replicated field sites across the United States during the 2006 growing season and across Chile during the 2006-2007 growing season. The U.S. field trials were conducted under typical agronomic practices, whereas the Chilean field trials incorporated a strip-plot design that included well-watered and water-limited treatments. Results demonstrated that levels of the components analyzed were comparable between MON 87460, the conventional control, and the commercially available corn hybrids.

Compositional equivalence of insect-protected glyphosate-tolerant soybean MON 87701 × MON 89788 to conventional soybean extends across different world regions and multiple growing seasons.

The soybean product MON 87701 × MON 89788 expresses both the cry1Ac gene derived from Bacillus thuringiensis and the cp4 epsps (5-enolpyruvylshikimate-3-phosphate synthase) gene derived from Agrobacterium sp. strain CP4. Each biotechnology-derived trait confers specific benefits of insect resistance and glyphosate tolerance, respectively. The purpose of this study was to compare the composition of seed and forage from this combined-trait product to those of conventional soybean grown in geographically and climatically distinct regions. Field trials were conducted in the United States during the 2007 growing season, in Argentina during the 2007-2008 growing season, and in the northern and southern soybean regions of Brazil during the 2007-2008 and 2008-2009 growing seasons. Results demonstrated that the compositional equivalence of MON 87701 × MON 89788 to the conventional soybean extended across all regions and growing seasons. Further evaluation of the data showed that natural variation (region and growing season) contributed more to compositional variability in soybean, particularly for such components as isoflavones, fatty acids, and vitamin E, than transgene insertion.

Compositional Variability in Conventional and Glyphosate-Tolerant Soybean (Glycine max L.) Varieties Grown in Different Regions in Brazil

The compositions of a diverse range of commercially available conventional and genetically modified (GM; glyphosate-tolerant) soybean varieties from maturity groups 8 and 5, respectively, grown in the northern and southern soybean regions of Brazil during the 2007-2008 and 2008-2009 growing seasons were compared. Compositional analyses included measurement of essential macro- and micronutrients, antinutrients, and selected secondary metabolites in harvested seed as well as measurement of proximates in both forage and harvested seed. Statistical comparisons utilized a mixed analysis of variance model to evaluate the relative contributions of growing season, soybean growing region, production site, phenotype (GM or conventional), and variety. The study highlighted extensive variability in the overall data set particularly for components such as fatty acids, vitamin E, and isoflavones. There were few differences between the GM and non-GM populations, and most of the variability in the data set could be attributed to regional and variety differences. Overall, the results were consistent with the expanding literature on the lack of any meaningful impact of transgene insertion on crop composition.

Stability in the composition equivalence of grain from insect-protected maize and seed from glyphosate-tolerant soybean to conventional counterparts over multiple seasons, locations, and breeding germplasms.

Insect-protected maize MON 810 and Roundup Ready soybean 40-3-2 represent major milestones in the adoption of genetically modified (GM) crops to enhance agricultural productivity. This study provides an assessment of the compositional stability of these products over multiple seasons, multiple germplasms, and diverse geographies encompassing North, Central, and South America and Europe. The compositional assessment evaluated levels of proximates in MON 810 and proximates, antinutrients, and isoflavones in 40-3-2. The means and range values for component levels in the GM crops and their conventional comparators were consistently similar to each other within each corresponding year from 2000 to 2009. To our knowledge, this study represents the first meta-analysis of comparative composition assessments of GM products. This approach, combined with graphical approaches, provided an effective summary of the overall data set and confirmed the continued compositional equivalence of these important crops to their conventional counterparts over time.

Opportunities and surprises in crops modified by transgenic technology: metabolic engineering of benzylisoquinoline alkaloid, gossypol and lysine biosynthetic pathways

The development of new or improved traits in plants, whether that is through traditional genetic modification and selection or through transgenic technologies, is associated with the potential risk of unintended changes with harmful or unacceptable consequences. The greater definition and precision of transgenic modification and the regulatory oversight of such technology may, however, confer advantages in safety and efficacy. This bears considerable relevance to the use of transgenic-based metabolic engineering in agricultural trait development. Metabolic engineering seeks to modify the amounts or chemical structures within selected biosynthetic routes without introducing inadvertent effects on other metabolic pathways. Examples discussed here include attempts to; (i) modify benzylisoquinoline alkaloid biosynthesis in poppy, (ii) improve the nutritional value of maize by increasing levels of free lysine, and (iii) increase the nutritional value of cottonseed by eliminating gossypol production. Clearly, evaluation of the efficacy (and unintended consequences) of such approaches is vital. A role for metabolomics in the compositional and metabolite analyses of new plant varieties derived from transgenic-based metabolic engineering is discussed. Major themes discussed in this review include; (i) the heightened level of scrutiny associated with genetically modified (GM) crop evaluations has markedly contributed to the safety in the adoption of transgenic technology, and (ii) the nature of any introduced trait may prove more relevant to safety assessments than the means by which the trait is introduced.